Expression of differentiation-associated keratins ina the skin is under transcriptional control. Abnormalities in the regulation of these genes probably occur in epidermal hyperplasia, dysplasia, and dyskeratotic cell formation, but detailed knowledge about the role that transcription factors play during either normal or abnormal differentiation does not yet exist. This is a proposal to study three C/EBP's (CAAT-enhancer binding proteins), transcription factors which appear to play a pivotal role in growth arrest and differentiation. CHOP (C/EBP-homologous protein) is a newly-described C/EBP that is inducible by UV light, and has been implicated in cancer. CHOP, and athe differentiation-associated proteins C/EBPalpha and C/EBPbeta, are all preferentially expressed in differentiating keratinocytes. Regulation of a prototype differentiation-related keratin gene (K10), which contains a c?EBP binding site, will be investigated. In normal epidermal differentiation, keratinocytes progress upward in an orderly manner, passing from a proliferating basal layer, through the spinous layer (at which point the k10 gene is activated), and terminating via apoptosis int he uppermost layers. Much of this program can ce recapitulated in murine BALB/MK keratinocytes, the model in which binding of the C/EBP's to the k10 gene promoter and subsequent transactivation of the k10 gene will be studied. Temporal and spatial patterns of expression of the transcription factors during calcium-induced differentiation will be defined and correlated with the status of cellular proliferation, differentiation, and apoptosis. A variety of DNA-protein binding assays and gene-transfection techniques will be used to assess the physical and functional interactions between the transcription factors and the k10 gene promoter in vitro and in vivo. Because C/EBP's are expressed in the skin, the proposed project will not only provide useful information about C/EBP- mediated regulation of the k10 gene in cultured keratinocytes, but will also lay the ground for future investigation of altered keratin expression n cutaneous disease states.